The present application relates to nucleic acid quantification methods, and microchips for nucleic acid amplification reaction. Specifically, the present application concerns nucleic acid quantification methods for conveniently measuring the approximate amount of the detection target nucleic acid chain contained in a sample.
Nucleic acid amplification methods such as PCR (Polymerase Chain Reaction) have been used in many applications, including the diagnoses of infections and hereditary diseases, gene expression level analyses, and cloning. Real-time nucleic acid amplification methods that measure the amplified amount of the detection target nucleic acid chain in real time based on the increased fluorescence intensity of the fluorescent dye or fluorescent dye-labeled fluorescent probe used for the measurement also have been used for the quantification of the original amount of the detection target nucleic acid chain.
Digital PCR, Proc. Natl. Acad. Sci. 1999, Vol. 96, p. 9236-9241 proposes a technique called “digital PCR” for accurately quantifying trace amounts of nucleic acid chain. In digital PCR, a sample containing the detection target nucleic acid chain is subjected to limiting dilution with a reaction solution, and dispensed in a plurality of reaction sites (wells) for PCR reaction. The number of wells that show fluorescence out of the amplification product is then counted using, for example, fluorescent probes. Because it can be assumed by limiting dilution that each well contains at most only one molecule (one copy) of the detection target nucleic acid chain, the copy number of the detection target nucleic acid chain contained in a sample can be quantified based on the number of wells showing fluorescence.
JP-A-2001-269196 proposes a digital PCR-based nucleic acid quantification method that makes use of a plate including several thousand to several million channels (reaction sites), each containing one copy of the detection target nucleic acid chain contained in a sample. With this method, it is considered possible to accurately quantify the copy number of the detection target nucleic acid chain, because the method does not involve a mismatch between the number of fluorescing wells and the copy number of the detection target nucleic acid chain, which occurs when one channel contains more than one copy of the detection target nucleic acid chain.